Modified oligonucleotides are of great value in molecular biological research and in applications such as anti-viral therapy. Modified oligonucleotides which can block RNA translation, and are nuclease resistant, are useful as antisense reagents. One such modification is the alkylphosphonate internucleotide linkage.
There are a number of properties associated with oligonucleotides having alkylphosphonate internucleoside linkages that are superior to native phosphodiester internucleotide linkages. The majority of research in this area has been with methylphosphonates (Miller et al., Antisense Research and Applications, 1993, pp. 189-203, Crooke, S. T. and Lebleu, B. (eds) CRC Press, Boca Raton). Oligonucleotides having methylphosphonate internucleotide linkages are isosteric to natural phosphate groups, have non-ionic backbones, are resistant to nucleases, have weak interactions with plasma proteins and can enter cells by passive diffusion or through absorptive endocytosis. Oligonucleotides having methylphosphonate internucleotide linkages are not substrates for RNase H when duplexed with RNA. When a chimeric oligonucleotide is prepared having a 2′-deoxy region flanked on either side by methylphosphonate regions, the resulting oligonucleotide is able to withstand intracellular nuclease degradation until bound to a target RNA. Upon formation of an oligonucleotide/target RNA duplex the 2′-deoxy region becomes a substrate for RNase H and thus can give rise to a terminating event.
In addition to chemical modifications of the internucleoside linkage, the chirality of each phosphorus containing internucleoside linkage also effects the ability of a modified oligonucleotide to bind to RNA. The standard oxidation of a phosphite to a phosphate internucleotide linkages results in attack on either side of the phosphorous atom resulting in Rp and Sp diastereomers. Modified phosphorus oligonucleotides are racemic mixtures having 2n isomers, with n equal to the number of intersugar linkages in the oligonucleotide. Thus, a 15-mer modified oligonucleotide, containing 14 asymmetric centers has 214 or 16,384 diastereomers. In view of this, in a racemic mixture, only a small percentage of the oligonucleotides are likely to specifically hybridize to a target mRNA or DNA with optimal affinity.
Backbone chirality may also affect the susceptibility of an oligonucleotide-RNA heteroduplex to RNase H activity. The ability to serve as a template for RNase H has significant therapeutic implications since it has been suggested that RNase H causes cleavage of the RNA component in an RNA-DNA oligonucleotide heteroduplex. For a variety of catalytic reactions, hydrolysis of the phosphodiester backbone of nucleic acids proceeds by a stereospecific mechanism (an in-line mechanism) and inversion of configuration. Therefore, there may be only a small percentage of oligonucleotides in a racemic mixture that contain the correct chirality for maximum hybridization efficiency and termination of translation. Thus, increasing the percentage of internucleotide linkages that are diastereomerically enriched that can serve as substrates for RNase H in a heteroduplex will likely lead to a more efficacious compound for antisense and other oligonucleotide therapies.
Oligonucleotides having pure Rp or Sp methylphosphonate internucleotide linkages have been prepared by a number of routes. The separation of a mixture of H-phosphonate diastereomers into pure Rp and Sp stereoisomers followed by alkylation with retention of configuration has given the corresponding pure Sp or Rp methylphosphonate (Seela, F., Kretschmer, U., J. Org Chem., 1991, 56, 3861-3869). Another method requires the separation of a mixture of Rp and Sp 5′-O-protected-2′-O-deoxynucleosyl-3′-O-methylphosphinate (International Patent Application PCT/US93/06251, entitled “Trivalent Synthesis of Oligonucleotides Containing Stereospecific Alkylphosphonates and Arylphosphonates”, filed Jun. 30, 1993), 3′-O-methylphosphonate (Jaworskamaslanka et al., Antisense & Nucleic Acid Drug Development, 1997, 7, 23-30), or -3′-O-methylphosphonothioate (International Patent Application PCT/US93/06277, entitled “Pentavalent synthesis of Oligonucleotides Containing Stereospecific Alkylphosphonates and Arylphosphonates”, filed Jun. 30, 1993) before their stereoselective incorporation in the oligonucleotide.
The incorporation of alkylphosphonate internucleoside linkages into modified oligonucleotide has been limited to only a few groups. The primary group that has been used is the methylphosphonate group. The use of benzylphosphonate internucleotide linkages is another group that has been synthesized (Eisenhardt et al., Nucleosides & Nucleotides, 1997, 16, 1669-1672).
The synthesis of a T—T dimer having a phosphorothioate internucleoside linkage has been previously reported using chiral auxiliary indole-oxazaphosphorine with 3′-O-TBDPS-thymidine in the presence of several equivalents of 1,8-diazabicyclo[5.40]undec-7-ene (DBU) (Wang, J. C., Just, G., Tetrahedron Lett., 1997, 38, 3797-3800). After removal of the chiral auxiliary and sulfurization, the T—T phosphorothioate dimer is isolated in very high diastereomeric excess.
There remains a need for improved methods for preparing alkylphosphonates. There is further need for methods for preparing alkylphosphonates that are substantially diastereomerically pure or enriched. The present invention is directed to these, as well as other, important ends.